Somatic Diversification of Immunoglobulin Genes in Galt

描述（由申请人提供）：肠道共生菌群是脊椎动物淋巴组织和粘膜免疫发育所必需的。然而，人们对这些过程发生的机制知之甚少。先前的家兔研究表明，在免疫前抗体库的形成过程中，肠道共生菌群也需要Ig基因的体细胞超突变（SHM）和基因转化（GC），以及B细胞的选择，这两者都发生在肠道相关淋巴组织（GALT）中。通过将选择的菌株引入无菌GALT，我们发现一些但不是所有的共生生物都具有诱导GALT发育和Ig基因SHM和GC的能力。我们发现两种共生微生物，脆弱芽孢杆菌和枯草芽孢杆菌，共同诱导这些过程；然而，单独引入时，这两种生物都不会引起这些过程。本研究的目的是确定这些细菌诱导GALT发育和Ig基因SHM和GC的机制，以及为什么需要两种细菌。第一个具体目标是确定细菌如何进入宿主，以及卵泡诱导细菌和非卵泡诱导细菌的差异是否存在于它们如何被GALT上皮吸收。为了验证这一点，实验包括将细菌引入无菌阑尾，然后进行原位杂交和免疫荧光来确定细菌定位在哪些细胞中。下一个具体目标是利用重组腺病毒表达的可溶性受体，研究树突状细胞（DC）是否通过分泌B细胞生长因子（BAFF）或抑制DC在体内的经上皮迁移来促进GALT的发育和Ig基因的多样化。在Aim 3中，对T细胞帮助的需求将通过抑制T细胞与可溶性CTLA4的激活和B-T细胞与可溶性CD40的相互作用来研究，两者都是在重组腺病毒中产生的。在Aim 4中，实验旨在寻找诱导GALT发展和一抗库体细胞多样化的B细胞超抗原。这些实验很重要，因为肠道内的细菌细胞比人体所有其他细胞的总和多10倍，但我们对这些微生物如何影响人类健康和过敏、炎症性肠病等疾病知之甚少。

DESCRIPTION (provided by applicant): The intestinal commensal microbiota is required for development of lymphoid tissues and mucosal immunity in vertebrates. However, little is known of the mechanism by which these processes occur. Previous work in rabbits has demonstrated that the intestinal commensal microbiota is also required for somatic hypermutation (SHM) and gene conversion (GC) of Ig genes during development of the preimmune antibody repertoire, and for B cell selection, both of which occur in gut-associated lymphoid tissues (GALT). By introducing select bacterial strains into sterile GALT, we found that some but not all commensal organisms have the capacity to induce GALT development and SHM and GC of Ig genes. We identified two commensal microorganisms, B. fragilis and B. subtilis, that together, induced these processes; however, introduced singly, neither organism induced these processes. The goal of this study is to determine the mechanism by which these bacteria induce GALT development and SHM and GC of Ig genes, and why two bacteria are required. The first specific aim is to determine how bacteria enter the host, and if the difference in follicle-inducing bacteria and non-follicle-inducing bacteria resides in how they are taken up across the epithelium of GALT. Experiments to test this include introducing bacteria into germfree appendix and then performing in situ hybridization and immunofluorescence to determine in which cells the bacteria are localized. The next specific aim, by using soluble receptors expressed in recombinant adenovirus, will investigate whether dendritic cells (DC) contribute to GALT development and Ig gene diversification by secretion of the B cell growth factor, BAFF, or by inhibiting the transepithelial migration of DC in vivo. In Aim 3, the requirement for T cell help will be investigated by inhibiting the activation of T cells with soluble CTLA4 and B-T cell interactions with soluble CD40, both produced in recombinant adenovirus. In Aim 4, experiments are designed to search for a B cell superantigen that induces GALT development and somatic diversification of the primary antibody repertoire. These experiments are important because the lumen of the intestine contains 10X more bacterial cells than all other cells of the human body combined, and yet we know little about how these organisms contribute to human health and diseases such as allergy and inflammatory bowel disease.